This invention relates to plant hormones, and in particular to cytokinins. Aspects of the invention include a purified zeatin O-glucosyltransferase enzyme, a purified zeatin O-xylosyltransferase enzyme, nucleic acid molecules encoding these enzymes, and vectors containing all or a portion of the nucleic acid molecules. Transgenic cells and transgenic plants having modified zeatin O-glucosyltransferase and/or modified zeatin O-xylosyltransferase activity are also provided. The invention also relates to altered plant traits in general, and seed development and yield in particular, resulting from the modification of zeatin O-glucosyltransferase and/or zeatin O-xylosyltransferase activity in plants.
Cytokinins are plant hormones that mediate cell division and development. This group of hormones was discovered by Miller et al. (1955, 1956) with the identification of the first synthetic cytokinin, kinetin. The first naturally occurring cytokinin, zeatin, was discovered by Letham (1963) in corn and the structure of zeatin was determined by Shaw and Wilson (1964). Zeatin is the most active and ubiquitous cytokinin in all plant species examined. Other naturally occurring cytokinins are structurally related to zeatin (Shaw, 1994).
The critical importance of cytokinins in plant development was illustrated by the classic tissue culture experiments of Skoog et al. (1965). These experiments established that plant cell division requires cytokinin. Furthermore, the ratio of cytokinins to auxins (another group of plant hormones) was shown to determine whether undifferentiated plant cells will develop into shoots (high cytokinin to auxin), or roots (low cytokinin to auxin) or continue to proliferate as callus tissues (intermediate cytokinin to auxin ratio). Thereafter, cytokinin was found to be involved in every phase of plant growth (Mok 1994). In general, cytokinins have growth promoting effects, from seed germination and shoot development to retarding senescence and increasing fruit and seed set.
The effects of cytokinins in controlling plant growth have been extensively utilized in plant tissue culture to micropropagate and clone plants and to regenerate whole plants from cells of many species (Krikorian 1995). In fact, the application of cytokinins in vitro contributes significantly to advances in plant biotechnology. In agricultural applications, external applications of cytokinins on whole plants are used to obtain enhanced fruit set and grain yield of food crops and longer shelf life of ornamentals (Hradecka and Petr 1992; Karanov et al. 1992; Lewis et al. 1996; Minana et al. 1989).
Cytokinins are converted to various metabolites in plant tissues (Jameson 1994). For example, the metabolites of zeatin include O-xylosylzeatin, O-glucosylzeatin, N-glucosylzeatin, zeatin riboside and zeatin nucleotides. The precise functions of these metabolites are still uncertain. However, some of these metabolites may be the stored or the transported form of the active compound, zeatin. The O-glucoside of zeatin may be such a metabolite (Badenoch-Jones 1996). It was first discovered by Letham et al. (1976) and has been found in all crops examined including corn, beans, poplar and soybean. As O-glucosylzeatin can be readily converted back to its active form, zeatin, by the removal of the glucose moiety (via the action of the widespread xcex2-glucosidase enzymes), it is considered a reversible reserve of active cytokinin (Brzobohaty et al. 1993). Also, O-glucosylzeatin is resistant to attack by cytokinin oxidases (McGaw and Horgan 1983) which degrade the parent compound, zeatin. Therefore, O-glucosylzeatin may be important in cytokinin action by serving as a remobilizable reserve and as a resistant form of zeatin.
The enzyme catalyzing the formation of zeatin to O-glucosylzeatin is termed zeatin O-glucosyltransferase. It was first purified and characterized in the inventor""s laboratory (Dixon et al. 1989) from lima beans (Phaseolus lunatus) as part of a continuing effort to study the metabolism of zeatin (Mok and Martin 1994). The isolation of the enzyme was followed by the generation of specific antibodies that recognize the enzyme (Martin et al. 1990). Enzyme assays and immunoblotting (western) analysis indicated that zeatin O-glucosyltransferase occurs in small developing seeds. As seed size is determined by cell number and cell size, both factors influenced by the level of cytokinins, the occurrence of zeatin O-glucosyltransferase in immature seeds suggests that the enzyme is likely to be of critical importance in determining the eventual seed yield.
Another enzyme that converts zeatin into a metabolite was identified in 1987. This enzyme was termed zeatin O-xylosyltransferase (Turner et al. 1987). The enzyme mediates the formation of O-xylosylzeatin from zeatin using UDPX as the sugar donor. The enzyme has similar properties as zeatin O-glucosyltransferase isolated from Phaseolus lunatus but can be distinguished by substrate specificity and charge (Dixon et al. 1989). Briefly, the O-xylosyltransferase utilizes UDPX as the sugar donor while the O-glucosyltransferase uses both UDPG and UDPX but with much greater affinity to UDPG. The O-xylosyltransferase also has higher negative charge than O-xylosyltransferase as determined by anion exchange column chromatography.
Plants having modified endogenous zeatin activity would be of significant agricultural importance. Such plants could be created through genetic engineering if the genes regulating zeatin were available. It is to such genes that the present invention is directed.
The present invention provides isolated plant nucleic acid molecules (cDNA and genomic sequences) encoding zeatin O-glucosyltransferase, a key enzyme in the regulation of zeatin activity in plants. The present invention also provides isolated plant nucleic acid molecules encoding O-xylosyltransferase, another key enzyme in the regulation of zeatin activity in plants.
In one embodiment, the zeatin O-glucosyltransferase nucleic acids disclosed are from the lima bean, Phaseolus lunatus. The open reading frame of these nucleic acid molecules encodes a polypeptide of 459 amino acids in length. This polypeptide is shown to have zeatin O-glucosyltransferase enzymatic activity i.e., it catalyzes the conversion of zeatin to O-glucosylzeatin. Accordingly, one aspect of the invention comprises isolated nucleic acid molecules encoding zeatin O-glucosyltransferase. Another aspect of the invention is the purified zeatin O-glucosyltransferase enzyme.
In another embodiment, the zeatin O-xylosyltransferase nucleic acids disclosed are from Phaseolus vulgaris. The open reading frame of these nucleic acid molecules encodes a polypeptide of 454 amino acids in length. This polypeptide is shown to have zeatin O-xylosyltransferase enzymatic activity i.e., it catalyzes the conversion of zeatin to O-xylosylzeatin. Accordingly, one aspect of the invention comprises isolated nucleic acid molecules encoding zeatin O-xylosyltransferase. Another aspect of the invention is the purified zeatin O-xylosyltransferase enzyme.
Also encompassed within the scope of this invention are transformation vectors which include at least a portion of the disclosed nucleic acid sequences. Such vectors may be transformed into plants to produce transgenic plants with modified zeatin O-glucosyltransferase activity and/or modified O-xylosyltransferase activity. Depending on the particular sequence incorporated into the vector, transformation with the zeatin O-glucosyltransferase cDNA and/or the O-xylosyltransferase cDNA, genes or derivatives thereof may be used to modify agronomically important traits, including the activity of zeatin in seeds, grain yield and seed germination rates. While all crop plants may benefit from such modified activity, it is anticipated that the invention will be particularly valuable in maize, wheat and legumes.
Typically, vectors used to modify zeatin O-glucosyltransferase activity and/or O-xylosyltransferase activity include regulatory sequences that are operably linked to the zeatin O-glucosyltransferase cDNA and/or zeatin O-xylosyltransferase cDNA, gene(s), or derivatives thereof. For example, zeatin O-glucosyltransferase activity may be modified in plants by introducing a transformation vector that includes a sense or antisense form of the disclosed cDNA operably linked to a high level constitutive promoter such as the 35S promoter of cauliflower mosaic virus. Transgenic plants transformed with such recombinant vectors and having modified zeatin O-glucosyltransferase activity and/or modified O-xylosyltransferase activity are part of the invention.
While the invention provides zeatin O-glucosyltransferase-encoding nucleic acids from Phaseolous lunatus, as well as zeatin O-xylosyltransferase-encoding nucleic acids from Phaseolous vulgaris, it additionally encompasses homologs, orthologs and derivatives of these sequences, as well as homologs, orthologs and variants of the zeatin O-glucosyltransferase polypeptide sequence and the zeatin O-xylosyltransferase polypeptide sequence. Thus, in one aspect of the invention, nucleic acid molecules that comprise specified regions of these sequences are provided. Exemplary of such nucleic acid molecules are oligonucleotides that are useful as probes or primers to detect and amplify zeatin O-glucosyltransferase-encoding nucleic acids and zeatin O-xylosyltransferase-encoding nucleic acids from other plant species. Such oligonucleotides are useful as hybridization probes or PCR primers, and typically comprise at least 15 consecutive bases of the disclosed sequences. In other embodiments, such oligonucleotides comprise longer regions of the disclosed sequences, such as at least 20, 25 or 30 consecutive nucleotides.
In another aspect, the invention provides compositions and methods for isolating nucleic acid sequences that have zeatin O-glucosyltransferase activity or zeatin O-xylosyltransferase activity from other plant species. Typically, such methods involve hybridizing probes or primers derived from the disclosed P. lunatus and P. vulgarissequences to nucleic acids obtained or derived from such other plant species.
Homologous and orthologous sequences to the P. lunatus zeatin O-glucosyltransferase, or the P. vulgaris zeatin O-xylosyltransferase nucleic acid and amino acid sequences share key functional and structural characteristics with the disclosed Phaseolous sequences. Functionally, such sequences encode (or comprise) a polypeptide that either catalyzes the O-glucosylation of zeatin or the O-xylosylation of zeatin. Structurally, such sequences share a specified structural relationship with the disclosed sequences. By way of example, in certain embodiments, homologous amino acid sequences have at least 70% sequence identity with the P. lunatus zeatin O-glucosyltransferase amino acid sequence or the P. vulgaris zeatin O-xylosyltransferase amino acid sequence. In other embodiments, homologous nucleic acid sequences hybridize under stringent conditions to the disclosed P. lunatus zeatin O-glucosyltransferase nucleic acid sequences or the P. vulgaris O-xylosyltransferase nucleic acid sequences.
Another aspect of the invention relates to the purified zeatin O-glucosyltransferase enzyme, and the purified zeatin O-xylosyltransferase enzyme. Having provided nucleic acid molecules that encode these enzymes, the invention facilitates the expression of zeatin O-glucosyltransferase and/or zeatin O-xylosyltransferase in heterologous systems, including E. coli, yeast and baculovirus expression systems. Thus, the invention permits the large scale production of the enzymes for agricultural and other applications.
In another aspect of the invention the promoter sequence of the P. lunatus zeatin O-glucosyltransferase gene is disclosed. This promoter sequence confers seed-specific expression, and may be used to express a variety of nucleic acids in a seed-specific manner.